Method for obtaining novel lutein-based formulations

ABSTRACT

The present invention describes a method of preparation of formulations of microcrystalline lutein, particularly in the form of esters, which are resistant to oxidation and are soluble in hydrophilic and/or lipophilic media. For these formulations, the esters of lutein are mixed with antioxidants, vegetable oils and/or organic solvents, and this initial mixture is submitted to various stages depending on the type of final formulation required. These formulations are suitable for direct application as colourants in the pharmaceutical, food and cosmetics fields. They can also be used as diet supplements.

FIELD OF THE INVENTION

The present invention relates to a new methodology for production oflutein formulations, basically esters of lutein with various fattyacids, starting from any natural or synthetic source, which impart ahigh added value to these molecules, since they make it possible toobtain stabilized preparations thereof for direct application in thefoodstuffs, pharmaceutical and cosmetics fields.

STATE OF THE ART

Traditionally, the carotenoids have been regarded as plant pigments. Infact they occur in all green tissues in the form of photosyntheticpigment-protein complexes within the chloroplasts. Despite the fact thatthe typical yellow to red colour of the carotenoids is masked by thegreen colour of the chlorophylls, the typical colouration imparted bythe carotenoids can be observed in the leaves of many trees in theautumn, when the chlorophyll decomposes, and the xanthophylls areesterified by mixtures of fatty acids. With few exceptions, thecarotenoids present in the majority of the leaves of all species areP,P-carotene, lutein, violaxanthin and neoxanthin. Of course, smallquantities of other carotenoids may also be encountered, such asβ,ε-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin, lutein5,6-epoxide and lactucaxanthin. Many flowers or fruits (tomato, orange,peppers, marigold, etc.), displaying a colour range from yellow to red,owe their colouration to the carotenoids located in their chromoplasts,and are often present in the form esterified by fatty acids (G. Britton,S. Liaaen-Jensen, H. Pfander, Carotenoids, Volume 1A: Isolation andAnalysis, 201, Publ. Birkhäuser, 1995).

The carotenoids can be divided into two classes: pure hydrocarbons,called carotenes, which include compounds such as β-carotene,α-carotene, γ-carotene or lycopene and xanthophylls, molecules thatcontain oxygenated functions, examples of this type being asthaxanthin,capsanthin, cantaxanthin or lutein. The two groups of compounds behavedifferently as regards their physicochemical properties and solubilityin organic solvents.

All these compounds play an important role in the human diet, and theirproperties as antioxidants for the prevention of cancer and other humandiseases and as precursors of vitamin A have been investigatedextensively. Furthermore, owing to their yellow to red colouration, thecarotenoids are used as a food supplement and colourant in margarine,butter, oils, soups), sauces, etc. (Ninet et al., Microbial Technology,2nd Edn, Vol. 1, 529-544 (1979), Academic Press NY, Eds. Peppler H. J.and Perlman D.).

Lutein, (3R, 3′R, 6′R)-β,ε-carotene-3,3′-diol, is a carotenoid belongingto the group of the xanthophylls or carotenoids with oxygenatedfunctions. It is a polyunsaturated asymmetric molecule that consists ofa carbon skeleton similar to that of α-carotene ((6′R)-β,ε-carotene),but having a β hydroxyl at C-3 and an α hydroxyl at C-3′. Its empiricalformula is C₄₀H₅₆O₂ with a molecular weight of 568.85 and the followingmolecular formula:

In 1907, on the basis of combustion analysis, together with classicaldeterminations of molecular weight, the molecular formula C₄₀H₅₆O₂ wasproposed for a compound isolated from green leaves, which was called“xanthophyll” (R. Willstatter and W. Mieg, Liebig's Ann. Chem., 335, 1(1907)). Nevertheless, the formula C₄₀H₅₆O₂ for the lutein isolated fromegg yolk was postulated some years later (R. Willstatter and H. H.Escher, Z. Physiol. Chem., 76, 214 (1912)), and it was not known at thattime that lutein and the compound isolated previously from leaves andcalled “xanthophyll” were the same.

Up to that moment, all attempts to elucidate the molecular structures ofthe carotenoids by classical experiments of chemical degradation untilidentifiable fragments were found, had not met with success. The highlyunsaturated nature of the carotenoids was confirmed some years later(1928) by experiments of catalytic hydrogenation, and it was then thatthe term polyene was applied for the first time (L. Zechmeister, L. VonCholnoky and V. Vrabely, Ver. Deut. Chem. Ges., 61, 566 (1928)).Starting from this moment, a clear and direct relation was establishedbetween colour and the number of conjugated double bonds present inthese molecules (R. Kuhn and A. Winterstein, Helv. Chim. Acta, 11, 87;116; 123; 144 (1928), and R. Kuhn and A. Winterstein, Helv. Chim. Acta,12, 493; 899 (1929)).

The correct formula of lutein (or “xanthophyll”) was established byKarrer in studies based on reactions of oxidative degradation (P.Karrer, A. Zubrys and R. Morf, Helv. Chim. Acta, 16, 977 (1933)).

The instability of the carotenoids in crystalline form is well known,and one method of stabilizing them is to prepare oily dispersions.Moreover, it is thought that when the carotenoids are dispersed in oilthey are absorbed more easily by the body.

An alternative method for the stabilization of unstable compounds istheir microencapsulation in starch matrices.

Thus, patents U.S. Pat. No. 2,876,160, U.S. Pat. No. 2,827,452, U.S.Pat. No. 4,276,312 and U.S. Pat. No. 5,976,575 describe a considerableincrease in the stability of various compounds, including thecarotenoids, by encapsulating them in a starch matrix.

One of the main difficulties in using the carotenoids in the field ofcolourants is their zero solubility in water, since many of theirapplications take place in aqueous media. This problem of solubility ismentioned in document U.S. Pat. No. 3,998,753, and was solved bypreparing solutions of carotenoids in volatile organic solvents, such ashalogenated hydrocarbons, and emulsifying them with an aqueous solutionof sodium lauryl sulphate.

Document U.S. Pat. No. 5,364,563 describes a method of producing apreparation of carotenoids in powder form, which involves forming asuspension of a carotenoid in a high-boiling-point oil. The suspensionis superheated with steam for a maximum period of 30 seconds to form asolution of carotenoid in oil. Next, this solution is emulsified with anaqueous solution of a colloid and then the emulsion is spray-dried.

In general, in the state of the art we have not found formulations oflutein that are resistant to oxidation for prolonged periods of storageand, at the same time, are soluble in lipophilic or hydrophilic media,permitting their use as colourants for foodstuffs, pharmaceuticals andin cosmetics, for example, or as diet supplements. Most of thecommercial samples of lutein consist of extracts or oleoresins fromplants, which have inadequate stability owing to their limited contentof antioxidants. Moreover, these oleoresins are difficult to use inhydrophilic environments, owing to their zero solubility in water, sothat their use is limited to applications in lipophilic environments. Incontrast, our formulations exhibit high stability owing to theircontrolled content of antioxidants, and are perfectly applicable in bothhydrophilic and lipophilic environments.

BRIEF DESCRIPTION OF THE INVENTION

The invention describes a method of formulation, finishing or finalpresentation of lutein, related compounds (basically esters of luteinwith various fatty acids) or mixtures of both, obtained from any naturalor synthetic source, depending on their final application, whichconsists of premixing with antioxidants in the presence of oils and/ororganic solvents, in suitable proportions.

It is possible to obtain, according to this method:

-   -   A microcrystalline suspension of lutein and/or related        compounds, in vegetable oil; suitable for applications in        lipophilic environments.    -   CWD lutein (cold-water-dispersible lutein); suitable for        applications in hydrophilic environments.

Each variant of the method of preparation of formulations comprises thefollowing stages:

-   -   Microcrystalline suspension of lutein and/or related compounds        in vegetable oil:    -   Mixing of the vegetable oil with the active molecule and an        antioxidant.    -   Milling of the mixture. CWD lutein (cold-water-dispersible        lutein):    -   Molecular dissolution of lutein and/or related compounds in an        organic solvent, preferably in the presence of antioxidants or        vegetable oils or both    -   Emulsifying of the organic solution of the active molecule with        an aqueous solution of modified starches    -   Evaporation of the organic solvent and of the water until the        dry residue is obtained and the appropriate level of residual        solvents    -   Drying and finishing of the product.

The method described endows this molecule with stability that issufficiently high (longer than 6 months in suitable conditions ofpackaging) to prevent its oxidation during storage.

DETAILED DESCRIPTION OF THE INVENTION

A principal object of this invention is a method of preparation ofvarious formulations as a function of the characteristics of theapplication for which it is intended to use lutein and/or its relatedcompounds. The said method consists of premixing of microcrystallinelutein with antioxidants in the presence of oils and/or organicsolvents, in suitable proportions.

A first formulation, called microcrystalline suspension of lutein invegetable oil, consists of premixing the lutein molecule to beformulated, with a variable quantity of vegetable oil. A great varietyof vegetable oils can be used, and the commonest, but not the only ones,are sunflower oil, olive oil, corn oil, soya oil, cottonseed oil, etc.The dose of lutein and/or related compound will depend on the finalstrength it is desired to achieve, the commonest values beingsuspensions with a content of active principle between 5 and 60%,preferably between 10 and 30%. To increase the stability of the mixture,the usual liposoluble antioxidants are used, such as naturaltocopherols, and preferably D,L-alpha-tocopherol. The proportion of thiscompound varies between 0.2 and 15% relative to the weight of the activemolecule, preferably between 0.5 and 5%. So that the formulationscontaining lutein and/or related compounds have satisfactoryphysiological activity it is necessary to reduce the size of thecrystals. This is achieved with the usual milling systems applicable toliquid mixtures. A special object of this invention is ball mills thatpermit reduction of crystal size below 10 microns, preferably below 5microns and even more preferably below 2 microns, using microspheresbetween 0.5 and 0.75 mm in diameter. However, crystal size can vary inrelation to the particular application of the suspension, in each caseemploying suitable spheres and milling conditions. The crystal size willalso determine the rheological properties of the mixture, especially itsviscosity, which can also be adjusted depending on requirements.

These microcrystalline suspensions of lutein and/or related compounds inoil are suitable for applications in lipophilic environments.

A second formulation, called cold-water-dispersible (CWD) luteinformulation, is based on the dissolution of lutein and/or relatedcompounds in an organic solvent and their subsequent microencapsulationin modified starches. This invention will refer in particular to the useof food-grade solvents that are regarded as natural, such as acylesters, preferably. ethyl, propyl, isopropyl, butyl or isobutylacetates, which combine the reasonably high solubility for thecarotenoid components with compatibility as solvents included in theGroup of Class III of the ICH. These solvents are permitted both atnational and at community level, in both the pharmaceutical and thefoodstuffs fields (RDL12/04/90 and RDL16/10/96). According to the ICH,the content of residual solvents must be below 5000 ppm, preferablybelow 1000 ppm and more preferably below 100 ppm, always based on thedry matter of the liquid mixture. The concentration of lutein and/orrelated compounds in the organic solvent can vary between 1 and 50 g/l,preferably between 10 and 30 g/l. The temperature of dissolution canvary between room temperature and the boiling point of the solvent,preferably between 20 and 130° C. The fact that the percentage of cislutein is a function of the temperature/time relation in the operationof dissolution of the molecule in the organic solvent means that if wewish to obtain a product with a low content of this isomer, either a lowdissolution temperature will be used, or otherwise a very shortdissolution time. Thus, in order to achieve low levels of cis, and owingto the relatively low solubility of these compounds in solvents of thistype (acyl esters) at temperatures of the order of 20-40° C.,dissolution will preferably be effected between 70 and 130° C. for a fewseconds. It should be noted that the trans isomer is the natural isomer,and that there are differences in shade of colouration between the twoisomers. On the other hand, if the levels of cis isomer are notimportant, dissolution can be carried out without restriction on itsconditions rather than achievement of complete solubility at themolecular level. Alternatively, it is possible to use a solvent withgreater solubility for these molecules at relatively low temperatures(20-35° C.), such as chloroform, methylene chloride, THF, etc. In thiscase dissolution can be effected at low temperature (around 30° C.) forsome minutes, without any risk of forming cis isomers in excessivelyhigh proportions. To increase the stability of the final formulation, anantioxidant, or mixtures of several antioxidants, preferably such astocopherol, ascorbyl palmitate, etc., each of them in a proportionbetween 1 and 30%, preferably between 10 and 20%, relative to the weightof the active molecule, are dissolved together with the lutein and/orrelated compounds in the organic solvent. It is also possible toincorporate vegetable oil in the mixture, i.e. sunflower oil, olive oil,corn oil, soya oil, cottonseed oil, etc., with the aim of promoting thedissolution of the lutein and/or related compounds, and giving thepreparation additional stability. The lutein/oil ratio can vary between10/1 and 1/10.

The solution of the active molecule thus obtained is mixed andemulsified with an aqueous solution containing an emulsifying agent, forexample modified starch, more concretely esters derived from starch,preferably octenyl succinates derived from starch of various molecularweights, particularly, but not exclusively, Purity Gum 2000® fromNational Starch or Cleargum CO 01® from Roquette, and amicroencapsulating agent, formed for example from modified starch, moreconcretely esters derived from starch, preferably octenyl succinatesderived from starch of various molecular weights, particularly, but notexclusively, Hi Cap 100® or Capsul® from National Starch. The mixingratio of the emulsifying agent and the microencapsulating agent can varybetween 5/95 and 95/5, preferably between 25/75 and 75/25, and morepreferably between 40/60 and 60/40. The water content of each of thecomponents of the mixture of emulsifying agent and microencapsulatingagent is variable, and can be between 1 and 30%, preferably between 5and 20%, and more preferably 10%. The mixture of aqueous and organicphases is emulsified and the emulsion obtained is homogenized usingpressure-difference homogenization systems of the Manton Gaulin orMicrofluidizer type, which are commonly used, and preferably byhomogenization by tangential friction, for example with an emulsifier ofthe Ultraturrax type, for a time that varies according to the energysupplied by the equipment and the volume of mixture to be emulsified,with the aim of obtaining an average micelle size below 10 microns,preferably below 2 microns and more preferably between 0.1 and 1 micron.

Once the emulsion has formed, evaporation of the organic solvent iseffected, preferably by vacuum distillation at a temperature below 50°C. As evaporation of the solvent takes place, microcrystallization ofthe active molecule occurs in the starch matrix. Once the solvent hasevaporated, evaporation is continued, with successive additions of wateruntil a content of residual solvents is obtained that meets thespecifications for maximum concentration stipulated in the legislation,and a dry residue that is suitable for the type of drying that is to beapplied to this liquid mixture. Suitable values of dry matter of thesuspension of microencapsulated lutein and/or related compounds arebetween 1 and 30%, preferably between 10 and 25%.

It is found, in accordance with the present invention, that both themethod of drying by high-temperature spraying (atomization) and themethod of fluidized-bed spraying (granulation) are suitable for dryingthe aqueous suspension of active molecule obtained. Another alternativewould be freeze-drying.

According to the method of drying by atomization, suitable inlettemperatures of the drying air would be between 100 and 200° C. whereasthe outlet temperatures would be between 60 and 120° C. The atomizedproduct has a particle size between 10 and 100 microns. In order toincrease the particle size and thus reduce the available surface area,and hence increase the oxidation stability of the product, the atomizedproduct can be submitted to a finishing process, consisting ofagglomeration by spraying a solution of one of the modified starchesused in the formulation, or of the actual suspension ofmicroencapsulated active molecule within a fluidized bed of the saidatomized product, making it possible to reach particle sizes in therange 50-500 microns, and preferably in the range 200-300 microns.

The granulation method involves the use of a fluidized-bed granulator inwhich seed material is placed, which can be a typical inert material,such as particles of sugar, or fine powder of the actual material to bedried, obtained in previous granulation operations or in a spray-dryingoperation. The particles are kept in motion by means of air, and thetemperature of the bed is maintained between 30 and 90° C., preferablybetween 50 and 80° C. The suspension of lutein and/or related moleculesis sprayed by means of air preheated to, a temperature between 20 and140° C. within the fluidized bed, at a velocity that ensures that theparticles to be coated are not wetted excessively and do not form lumps.The granulated product has a particle size between 100 and 2000 microns,preferably between 100 and 800 microns, and more preferably between 100and 300 microns.

On completion of the spray-drying stage by one or other method, as wellas optional agglomeration, the particles obtained can be submitted to afinishing process by coating. This coating can be effected withapproximately 0.5-10% by dry weight, of aqueous solutions of sugars oreven starches.

EXAMPLE 1

A laboratory ball mill of the Minizeta 003 type from Netzsch is loadedwith—in this order—microspheres 0.5-0.75 mm in diameter, 30 g ofsunflower oil (Koipe), 0.08 g of D,L-alpha-tocopherol (Merck) and 20 gof lutein eter Xantopina Plus (Bioquimex), which has an equivalentlutein content of 40%. The mixture was milled at 3000 rpm for 5 minutes,obtaining 45 g of an orange-coloured, viscous liquid. Spectrophotometricanalysis of the oily suspension revealed a lutein content of 15%. Thecrystal size was less than 10 microns.

EXAMPLE 2

20 g of lutein ester Xantopina Plus (Bioquimex), which has an equivalentlutein content of 40%, was resuspended in 410 ml of isobutyl acetate,and 0.8 g of D,L-alpha-tocopherol (Merck) was added. The mixture washeated to boiling (114° C.) for 2 minutes, achieving completedissolution of the solid. As a parallel operation, 26.65 g of Hi Cap100® (National Starch) and 26.65 g of Purity Gum 2000® (National Starch)were dissolved in 325 ml of demineralized water. The hot organic phasewas emulsified for 10 minutes in one stage over the aqueous phase usingan Ultraturrax emulsifier from IKA, obtaining an average micelle size of0.4 micron, measured with a Coulter LS230 analyser. The emulsion wastransferred to a vacuum distillation system, adding 600 ml of water, sothat the 410 ml of isobutyl acetate was evaporated with approximately700 ml of water. 225 g of liquid formulation (25.9% of dry matter) wasobtained with an equivalent lutein content of 2.6% (10.1% based on thedry mass). This liquid formulation was dried in an Aeromatic AGlaboratory granulator, employing an inlet gas temperature of 90° C. andachieving a product temperature of 70° C., obtaining an orange-colouredpowder with an equivalent lutein content of 9.7% and a water content of2.6%.

EXAMPLE 3

20 g of lutein ester Xantopina Plus (Bioquimex), which has an equivalentlutein content of 40%, was resuspended in 410 ml of isobutyl acetate,and 0.8 g of D,L-alpha-tocopherol (Merck), 1.6 g of ascorbyl palmitate(Merck) and 8 g of sunflower oil (Koipe) were added. The mixture washeated to boiling (114° C.) for 2 minutes, achieving completedissolution of the solid. As a parallel operation, 21.5 g of Hi Cap 100®(National Starch) and 21.5 g of Purity Gum 2000® (National Starch) weredissolved in 325 ml of demineralized water. The hot organic phase wasemulsified for 10 minutes in one stage over the aqueous phase using anUltraturrax emulsifier from IKA, obtaining an average micelle size of0.5 micron, measured with a Coulter LS230 analyser. The emulsion wastransferred to a vacuum distillation system, adding 600 ml of water, sothat the 410 ml of isobutyl acetate was evaporated with approximately700 ml of water. 205 g of liquid formulation (25.0% of dry matter) wasobtained with an equivalent lutein content of 2.5% (10.0% based on thedry mass). This liquid formulation was dried in an Aeromatic AGlaboratory granulator, employing an inlet gas temperature of 90° C. andachieving a product temperature of 70° C., obtaining an orange-colouredpowder with an equivalent lutein content of 9.5% and a water content of3.0%.

EXAMPLE 4

20 g of lutein ester Xantopina Plus (Bioquimex), which has an equivalentlutein content of 40%, was resuspended in 500 ml of dichloromethane, and0.8 g of D,L-alpha-tocopherol (Merck) was added. The mixture was heatedat 35° C. for 5 minutes, achieving complete dissolution of the solid. Asa parallel operation, 26.65 g of Hi Cap 100® (National Starch) and 26.65g of Purity Gum 2000® (National Starch) were dissolved in 400 ml ofdemineralized water. The hot organic phase was emulsified for 10 minutesin one stage over the aqueous phase using an Ultraturrax emulsifier fromIKA, obtaining an average micelle size of 0.5 micron, measured with aCoulter LS230 analyser. The emulsion was transferred to a vacuumdistillation system, adding 600 ml of water, so that the 500 ml ofdichloromethane was evaporated with approximately 800 ml of water. 200 gof liquid formulation (26% of dry matter) was obtained with anequivalent lutein content of 2.6% (10.0% based on the dry mass). Thisliquid formulation was dried in an Aeromatic AG laboratory granulator,employing an inlet gas temperature of 90° C. and achieving a producttemperature of 70° C., obtaining an orange-coloured powder with anequivalent lutein content of 9.8% and a water content of 2.0%.

1. A process of obtaining a microcrystalline formulation based onlutein, its esters of fatty acids or mixtures thereof, derived from anysource, whether natural or synthetic, consisting in: a) dissolution ofthe lutein in a food-grade organic solvent in the presence ofantioxidants or vegetable oils or both, at temperatures ranging 30-130°C., depending on the solvent used, b) emulsifying and microencapsulatingthe organic solution obtained in the previous step with an aqueoussolution of modified starch by using homogenization means, c)evaporating the organic solvent and the water until a content ofresidual solvents suitable for food-grade commercialisation is obtainedand microcrystallization of lutein occurs d) drying and finishing.
 2. Aprocess according to claim 1, characterized in that preferablytocopherol or ascorbyl palmitate is used as the antioxidant, in aproportion from 0.2 to 30%, preferably in the range 10-20%.
 3. A processaccording to claim 1, characterized in that liposoluble antioxidantcompounds are used, in a proportion between 0.5 and 10%, based on theweight of lutein in the mixture.
 4. A process according to claim 1, inwhich the oil used is of plant origin, preferably sunflower oil, oliveoil, corn oil, cottonseed oil, peanut oil or soya oil.
 5. A processaccording to claim 1, characterized in that the organic solvent used ispreferably selected from the following: methylene chloride, chloroform,TMF, ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetateor isobutyl acetate.
 6. A process according to claim 1, wherein theshield of the evaporation step c) is a residual content of organicsolvent below 5000 ppm, preferably below 1000 ppm and more preferablybelow 100 ppm, relative to the dry matter of the suspension.
 7. Aprocess according to claim 1, characterized in that starch esters,preferably octenyl succinate derivatives of starch, are used as theemulsifying/microencapsulating agents.
 8. A process according to claim1, characterized in that drying of the liquid suspension is effected byatomization at temperatures in the range 100-200° C. for the inlet airand 60-120° C. for the outlet air.
 9. A process according to claim 1,characterized in that drying of the liquid suspension is effected byfluidized-bed spraying at temperatures, for the bed, in the range 30-90°C., and preferably 50-80° C., the suspension being sprayed over the saidbed with air preheated to 20-140° C.
 10. A process according to claim 1,characterized in that drying of the liquid suspension is effected byfreeze-drying.
 11. A process according to claim 1, characterized in thatfinishing consists of coating the particles with aqueous solutions ofvarious sugars, or of modified starches.
 12. A formulation obtainableaccording to the process of claim 1, characterized in that it consistsof granules of microcrystals of lutein and/or its esters of fatty acids,having the microcrystals an average size, measured as average micellesize, below 10 microns, preferably below 2 microns and more preferablybetween 0.1 and 1 micron, and with an average granule size in the range100-2000 microns, preferably in the range 100-800 microns and morepreferably in the range 100-300 microns.
 13. A formulation obtainableaccording to the process of claim 1, characterized in that it consistsof an atomized product of microcrystals of lutein and/or its esters offatty acids, having the microcrystals an average size, measured asaverage micelle size, below 10 microns, preferably below 2 microns andmore preferably between 0.1 and 1 micron, with an average particle sizeof the atomized product in the range 10-100 microns.
 14. A formulationobtainable according to the process of claim 1, characterized in that itconsists of an agglomerate of an atomized product of microcrystals oflutein and/or its esters of fatty acids, having the microcrystals anaverage size, measured as average micelle size, below 10 microns,preferably below 2 microns and more preferably between 0.1 and 1 micron,with an average agglomerate size in the range 50-500 microns, preferablyin the range 200-300 microns.
 15. A formulation according to claim 12,characterized in that it is coated with
 0. 5-10% by dry weight ofaqueous solutions of sugars or modified starch.
 16. Use of any of theformulation of claim 12, as colourants, especially in the food,pharmaceutical and cosmetics sectors.
 17. Use of any of the formulationsof claim 12 as diet supplements.